The commonly used means to differentiate between normal and non-normal states of a human subject using his or her biological materials are mainly those which have been used in the field of diagnostics. Most frequently used are those methods which target biomarkers in blood. It has been practiced in this field to comparatively measure the amount of a specific protein or a peptide that is less than 10,000 in molecular weight or, in the case of enzyme protein, enzyme activities in samples from normal (healthy) subjects and those from diseased individuals to help diagnosis. Here, prior to testing real samples, measurements are done on a fixed number each of samples from healthy controls and patients with certain disease with respect to the amount(s) or activity (activities) of single or multiple specific proteins or peptides and the ranges of abnormal and normal values are respectively determined. The sample to be evaluated is then analyzed by the same method and the resultant value is judged with respect to whether it is in normal or abnormal range.
In the actual measurements, the amount(s) of specified protein(s) or peptide(s) in test samples, as such or after dilution, are determined by the use of enzyme-linked immmunosorbent assay (ELISA) which uses a primary, or secondary, antibody labeled with an enzyme reacting with a substrate that yields a color upon reaction, chemiluminescent immunoassay (CLIA), radioimmunoassay (RIA) which uses a primary, or secondary, antibody labeled with a radioisotope, and, if the protein is an enzyme, the measurement of the activity of the enzyme by adding its substrate and determining the intensity of produced color, etc. These antibody-based methods are called as enzyme-, fluorescence- or radioisotope-labeled methods, respectively. In addition, there is a method where an enzyme reaction product derived from the corresponding substrate is determined by high performance liquid chromatography (HPLC). In further addition, there is a method where HPLC is combined with mass spectrometer, called LC-MS/MS, and there is a method called selected reaction monitoring (SRM)/multiple reaction monitoring (MRM) that utilizes LC-MS/MS. In another method to determine the concentration in a sample, it is appropriately pretreated, and separation of proteins or peptides is attained by 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and target protein or peptide is determined by silver staining, Coomassie blue staining or immunological staining (Western blotting) that uses an antibody to target protein or peptide. In still further addition, there is a method which utilizes mass spectrometry to determine the amount of target protein or peptide in samples fractionated by column chromatography. Instead of column chromatography, protein chips and magnetic beads may also be utilized for purpose of pretreatment.
Furthermore, these inventors have developed an immunoMS method, where target protein or peptide is captured by beads (including magnetic ones) with linked antibody to the protein or peptide, eluted from the beads, and determined by mass spectrometry. Further, intact proteins have been reported to be analyzed by mass spectrometry using above-mentioned methods after digestion with trypsin etc. (Patent Document 1). Here, intact target proteins are selected either by fractionation or by adsorption to an adsorbant specific to them and then determined by mass spectrometry.
Nonalcoholic fatty liver disease is abbreviated as NAFLD and patients with this disease, despite the fact that they have no drinking habit (less than 20 g daily), give histological findings characterized by hepatic fatty deposition reminiscent of those found in alcoholic hepatic damage. The disease caused viruses such as HCV or HBV or of autoimmune origin is excluded. The disease is regarded as a phenotype in the liver of metabolic syndrome accompanying obesity. NAFLD is divided into simple fatty liver and nonalcoholic steatohepatitis. The latter, abbreviated as NASH, is a progressive disease. NASH frequently accompanies fibrosis and has been known to often progress to hepatic cirrhosis and further to hepatic cancer. These features have attracted attention to this disease (Non-patent Document 1). Hereafter in this specification, simple fatty liver is called fatty liver.
Fatty liver is suspected in regular checkups from high levels of triglyceride in blood and diagnosed by abdominal ultrasonography and CT. Attention is warranted to the fact that 40% of fatty liver in non-drinker is accompanied by hepatic damage. Despite the observation that nonalcoholic fatty liver progresses to NASH, no convenient blood test is available for NASH.
While NASH is regarded as a severe type of NAFLD, routine blood chemistry, specifically the values of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) increase only slightly and the disease is often overlooked. Hence, it has been pointed out to be an important issue requiring resolution because there is no specific test method to detect it as in the case of fatty liver.